The invention relates to a new 1,3-propanediol monomer and polymers derived from these monomers. More specifically, polypropylene terephthalate has been produced from a 1,3-propanediol monomer prepared by bioconverting a fermentable carbon source directly to 1,3-propanediol using a single microorganism.
1,3-Propanediol is a monomer useful in the production of polyester fibers and in the manufacture of polyurethanes.
It has been known for over a century that 1,3-propanediol can be produced from the fermentation of glycerol. Bacterial strains able to produce 1,3-propanediol have been found, for example, in the groups Citrobacter, Clostridium, Enterobacter, Ilyobacter, Klebsiella, Lactobacillus, and Pelobacter. In each case studied, glycerol is converted to 1,3-propanediol in a two step, enzyme-catalyzed reaction sequence. In the first step, a dehydratase catalyzes the conversion of glycerol to 3-hydroxypropionaldehyde (3-HP) and water, Equation 1. In the second step, 3-HP is reduced to 1,3-propanediol by a NAD+-linked oxidoreductase, Equation 2. The 1,3-propanediol is not metabolized further and, as a result,
Glycerolxe2x86x923xe2x80x94HP+H2Oxe2x80x83xe2x80x83(Equation 1)
3xe2x80x94HP+NADH+H+xe2x86x921,3-Propanediol+NAD+xe2x80x83xe2x80x83(Equation 2)
accumulates in high concentration in the media. The overall reaction consumes a reducing equivalent in the form of a cofactor, reduced xcex2-nicotinamide adenine dinucleotide (NADH), which is oxidized to nicotinamide adenine dinucleotide (NAD+).
The production of 1,3-propanediol from glycerol is generally performed under anaerobic conditions using glycerol as the sole carbon source and in the absence of other exogenous reducing equivalent acceptors. Under these conditions in e.g., strains of Citrobacter, Clostridium, and Klebsiella, a parallel pathway for glycerol operates which first involves oxidation of glycerol to dihydroxyacetone (DHA) by a NAD+- (or NADP+-) linked glycerol dehydrogenase, Equation 3. The DHA, following phosphorylation to dihydroxyacetone phosphate (DHAP) by a DHA kinase (Equation 4),
xe2x80x83Glycerol+NAD+xe2x86x92DHA+NADH+H+xe2x80x83xe2x80x83(Equation 3)
DHA+ATPxe2x86x92DHAP+ADPxe2x80x83xe2x80x83(Equation 4)
becomes available for biosynthesis and for supporting ATP generation via e.g., glycolysis. In contrast to the 1,3-propanediol pathway, this pathway may provide carbon and energy to the cell and produces rather than consumes NADH.
In Klebsiella pneumoniae and Citrobacter freundii, the genes encoding the functionally linked activities of glycerol dehydratase (dhaB), 1,3-propanediol oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK) are encompassed by the dha regulon. The dha regulons from Citrobacter and Klebsiella have been expressed in Escherichia coli and have been shown to convert glycerol to 1,3-propanediol.
Although biological methods of both glycerol and 1,3-propanediol production are known, it has never been demonstrated that the entire process can be accomplished by a single organism.
Neither the chemical nor biological methods described above for the production of 1,3-propanediol is well suited for industrial scale production. This is because the chemical processes are energy intensive and the biological processes require glycerol, an expensive starting material. A method requiring low energy input and an inexpensive starting material is needed. A more desirable process would incorporate a microorganism that would have the ability to convert basic carbon sources, such as carbohydrates or sugars, to the desired 1,3-propanediol end-product.
There are several difficulties that are encountered when attempting to biologically produce 1,3-propanediol by a single organism from an inexpensive carbon substrate such as glucose or other sugars. The biological production of 1,3-propanediol requires glycerol as a substrate for a two-step sequential reaction in which a dehydratase enzyme (typically a coenzyme B12-dependent dehydratase) converts glycerol to an intermediate, 3-hydroxypropionaldehyde, which is then reduced to 1,3-propanediol by a NADH- (or NADPH) dependent oxidoreductase. The complexity of the cofactor requirements necessitates the use of a whole cell catalyst for an industrial process which utilizes this reaction sequence for the production of 1,3-propanediol. Furthermore, in order to make the process economically viable, a less expensive feedstock than glycerol or dihydroxyacetone is needed. Glucose and other carbohydrates are suitable substrates, but, as discussed above, are known to interfere with 1,3-propanediol production.
The present invention provides a 1,3-propanediol composition of matter produced by the process comprising the bioconversion of a carbon substrate, other than glycerol or dehydroxy acetone dihydroxyacetone, to 1,3-propanediol by a single microorganism having at least one gene that expresses a dehydratase enzyme by contacting said microorganism with said substrate.
The invention further provides a biosourced 1,3-propanediol composition of matter having a xcex413C of about xe2x88x9210.9 to about xe2x88x9215.4, preferably about xe2x88x9213.22 to about xe2x88x9214.54, and most preferably about xe2x88x9213.84 to about xe2x88x9213.92, and a fM 14C of about 1.04 to about 1.18, preferably about 1.106 to about 1.129, and most preferably about 1.111 to about 1.124.
Additionally the invention provides a polymer comprising at least two repeating units of biosourced 1,3-propanediol, characterized by a xcex413C of xe2x88x9210.74 to about xe2x88x9217.02, preferably about xe2x88x9213.22 to about xe2x88x9214.54, and most preferably about xe2x88x9213.84 to about xe2x88x9213.82 to about xe2x88x9213.94, and a fM 14C of about 1.003 to about 1.232, preferably about 1.106 to about 1.129, and most preferably about 1.111 to about 1.124.
In another embodiment, the invention provides a polymer comprising at least two repeating units of biosourced polypropylene terephthalate, characterized by a xcex413C of about xe2x88x9224.74 to about xe2x88x9224.88, and a fM 14C of about 0.299 to about 0.309 and a polymeric unit consisting of polypropylene terephthalate having a xcex413C of about xe2x88x9224.74 to about xe2x88x9224.88, and a fM 14C of about 0.299 to about 0.309.
In another embodiment the invention provides a method for identifying the presence of a biosourced 1,3-propanediol in a sample, the method comprising (a) purifying the 1,3-propanediol from the sample; and (b) determining the xcex413C and fM 14C characterizing the sample of step (a), wherein a xcex413C of about xe2x88x9210.9 to about xe2x88x9215.4 and a fM 14C of about 1.04 to about 1.18 indicates the presence of a biosourced 1,3-propanediol. Additionally, the specific source of biosourced carbon (e.g. glucose or gycerol) can be ascertained by dual carbon-isotopic analysis.
Finally, the invention provides an article of manufacture comprising the described composition produced by the process and in a form selected from the group consisting of a film, a fiber, a particle, and a molded article.